T cell receptor gene segment utilization by HLA-DR1-alloreactive T cell clones.

Transplantation of histoincompatible tissues leads to allograft rejection, which involves recognition of allogeneic MHC molecules by Ag-specific receptors expressed on T cells. The interaction of these molecules is highly specific yet poorly understood. We have investigated the relationship between TCR gene utilization and allo-MHC restriction patterns by using a one-way polymerase chain reaction to amplify the alpha- and beta-chain mRNA from a panel of 10 HLA-DR1-alloreactive T lymphocyte clones. Two previously unreported V alpha and five J alpha gene sequences were obtained. Although a few V alpha, V beta, and J alpha genes were utilized more than once, no correlation between TCR gene usage and DR1 alloreactivity was identified. At the sequence level, the presumed TCR alpha- and beta-chain CDR1 and CDR2 regions displayed limited diversity, whereas the CDR3 or junctional sequences were highly variable. Although most TCR probably interact with subtly different surface features of the DR1 alloantigen, we predict that TCR with similar CDR1 and CDR2 sequences would contact essentially identical regions of the DR1 molecule. The lack of sequence conservation in the junctional regions suggests that different endogenous peptides also may be recognized. Thus, alloreactive T cells may recognize not only allogeneic MHC molecules but perhaps also bound endogenous peptides.     

Modulation of in vitro myelopoiesis by alloreactive T cell clones

Regulatory effects of mixed lymphocyte culture (MLC)-derived CD4+ human T cell clones on granulocyte-macrophage colony (CFU-GM) formation by normal bone marrow (BM) were studied in an initial attempt to establish an in vitro model for the negative feedback control of myelopoiesis by alloactivated T cells. This is likely to be of clinical significance in the aberrant control of haematopoiesis during some cases of graft-versus-host disease (GVHD) after allogeneic BM transplantation. Whilst 5 such alloproliferative clones generally failed to suppress CFU-GM, the majority of clones with natural killer (NK)-like activity, or those with suppressive activity in MLC, regularly and strongly suppressed in this system, reinforcing the view that certain T cells may have potent negative regulatory effects on haematopoiesis.     

Detection of HLA-DR associated PLT determinants by alloreactive lymphocyte clones

This study deals with alloreactive T-cell clones which recognize cellular determinants associated with HLA-DR antigens. Two clones, CB55 and DS56, exhibited a PLT specificity that was perfectly associated with DR5. On the other hand, clones CB7, DS1 and HS1 showed PLT reactivity with approximately one-half of the DR5 positive cells and none of the DR5 negative cells, whereas clone MD4 largely reacted with the other half of DR5 positive cells. Another MLR culture generated two alloreactive clones DS6 and DS9 with PLT specificity for DR2. However, these clones did not respond to DR2 cells, which were also positive for the DR2-associated HLA-B7 and B18 antigens. Monoclonal antibody (mAb) inhibition studies showed heterogenous patterns, whereby monomorphic non-DR mAbs inhibited the DR2-associated PLT clones while the DR5-associated PLT clones were inhibited by different groups of anti-DR and non-DR mAbs. These observations suggest the existence of several lymphocyte-activating determinants associated with HLA-DR antigens. This diversity may be an important consideration in studies of the role of HLA-DR in immune mechanisms and transplant compatibility.     

Antigen-specific human T lymphocyte clones: mechanisms of inhibition of proliferative responses by xenoantiserum to human nonpolymorphic HLA-DR antigens

We studied the effects of xenoantiserum to human nonpolymorphic Ia-like antigens upon in vitro antigen-specific T cell proliferative responses in unfractionated PBL populations and at the monoclonal level. Our findings suggest that the xenoantiserum, although it inhibits the antigen-specific response of unfractionated PBL and allospecific T cell clones, does not inhibit the proliferative response to cloned influenza virus immune human T lymphocytes, and therefore may be mediating inhibition by dual mechanisms: direct inhibition of alloantigen recognition and induction of nonspecific suppression. Kinetic differences may explain these phenomena. In cocultivation experiments with a virus-specific clone, the RaIa antiserum appears to induce an OKT3+,8+,4-, radiosensitive regulatory subset of lymphocytes. When adoptively transferred, these induced cells inhibit the TLC response in an antigen-nonspecific and genetically nonrestricted manner. We discuss the various modes and levels of inhibition of antigen-specific proliferation by anti-Ia antisera and their multiple activities.     

T lymphocyte cloning from rejected human kidney allograft. Recognition repertoire of alloreactive T cell clones

We analyzed the recognition repertoire of 16 human alloreactive T cell clones (ATLC) derived from cells invading an irreversibly rejected kidney allograft. These clones, which specifically proliferated against the kidney donor B lymphoblastoid cell line, fell into two classes: CD4+ killers and CD8+ killers. Cytotoxic and proliferative activities of the ATLC were studied by using a panel of allogeneic cells sharing HLA specificities with kidney donor cells. Moreover, mAb recognizing monomorphic parts of HLA class I and class II molecules were used in blocking experiments of ATLC cytotoxicity. The results obtained from proliferative and cytotoxic assays were concordant. All ATLC investigated were directed against HLA molecules, and some clones were found to recognize HLA-B, -C, -DP, -DQ, or -DR products. All anti-HLA class I ATLC were CD8+, whereas both CD4+ and CD8+ ATLC were committed against HLA class II specificities. Nine of 16 ATLC were shown to react against serologically defined donor HLA determinants. These data indicate the recognition of HLA determinants in the course of an in vivo alloimmune response and particularly emphasize the role of HLA-C and DP loci products so far ignored in clinical transplantation.     

Allospecific human T cell lines and clones which mediate HLA-DR restricted helper activity

In order to analyze the immunoregulatory activity of allospecific human T cells, we have combined the techniques of limiting dilution culture and IL-2 dependent T cell growth to generate cloned alloreactive T cell lines (TCL). These TCL have been expanded in continuous culture for greater than 8 mo with retention of stable phenotypic and functional characteristics. For example, phenotypic analysis of alloproliferative TCL, utilizing a panel of monoclonal antibodies, demonstrate that these cultures are comprised of T cells belonging exclusively to the T3+ T4+ T8- "helper" or "inducer" T cell subset. Coculture of cloned alloproliferative TCL cells with a panel of allogeneic stimulators reveals that these clones are specifically reactive against the HLA-DRw1 determinant. Of greater interest, coculture of selected alloproliferative TCL cells with DRw1+ but not DRw1- B cells results in a vigorous polyclonal response as measured by the reverse hemolytic plaque assay. Although major histocompatibility complex restriction of this T-B interaction operates at the inductive level, help is at least partially unrestricted at the effector level as alloantigen-activated TCL cells provide demonstrable, although not maximal, help for DRw1- B cells.     

Glucocorticoid inhibition of lymphokine secretion by alloreactive T lymphocyte clones

The effect of glucocorticoids on lymphokine production by T lymphocytes was examined by using long-term alloreactive T cell clones that secreted one or more of the lymphokines interleukin 2 (IL 2), interferon-gamma, macrophage-activating factor (MAF), and colony-stimulating factor when stimulated by an antigen or a mitogen. Production of all of these four lymphokines was inhibited when glucocorticoids were added at physiologic concentrations (10(-8) to 10(-6) M) to clones stimulated with concanavalin A (Con A). Clones were heterogeneous with respect to their sensitivity to glucocorticoid inhibition of MAF production; cytolytic clones were generally more resistant than noncytolytic clones. The glucocorticoid dexamethasone (Dex) and an IL 2-containing supernatant exerted opposing effects on clonal MAF production. Kinetics experiments showed that Dex inhibited MAF production by reducing the rate of secretion without causing a compensatory increase in the duration of secretion, whereas the IL 2 source increased the rate and the total amount of MAF secretion. Dex abrogated the effect of IL 2. Inhibition by Dex was apparent from the earliest time of detectable MAF production (about 4 hr after stimulation) and increased with longer exposure until production ceased (12 to 24 hr). Pre-exposure and removal of Dex before Con A stimulation also inhibited MAF release. Effects of Dex on lymphokine secretion by clones could be dissociated from effects on their growth in response to stimulator cells and IL 2. Factor production by the 16 clones tested was inhibited to some degree. Proliferation, however, by two of these clones (both cytolytic) was unaffected by Dex, whereas proliferation of two noncytolytic clones was strongly inhibited even in the presence of a saturating dose of IL 2.     

Association of PLT specificity of alloreactive lymphocyte clones with HLA-DR,MB and MT determinants

The HLA-D region encodes for several serologically defined systems, including DR, MB, and MT. The antigens of MB and MT are strongly associated with two or more DR specificities. The purpose of this study was to determine the role of MB and MT antigens in lymphocyte alloactivation. A soft agar colony assay was used to generate alloreactive lymphocyte clones primed in mixed leukocyte culture against a stimulator who typed as HLA-DR4,-;MB3,-; MT3,-. In secondary primed lymphocyte typing (PLT) assays, several clones were identified with PLT specificities strongly associated with DR4, MB3, or MT3. The data suggest that HLA-D controls different lymphocyte-activating determinants associated with the serologically defined DR, MB, or MT antigens.     

IgE production by normal human B cells induced by alloreactive T cell clones is mediated by IL-4 and suppressed by IFN-gamma

Seven T cell clones were established from mixed leukocyte cultures in which PBMC from two healthy donors and from one patient suffering from the hyper-IgE syndrome were stimulated by the irradiated EBV-transformed B cell lines JY or UD53. Five of seven T cell clones, after activation by co-cultivation with JY or UD53 cells, induced a low degree of IgE production by normal blood B cells. In one experiment in which the normal B cells could activate the T cell clones directly, IgE production was also observed in the absence of the specific stimulator cells. IgE production was also obtained with supernatants of the T cell clones collected 4 to 5 days after activation by their specific stimulator cells. In addition, the supernatants induced IgG, IgA, and IgM synthesis. All seven clones produced variable concentrations of IL-4 and IFN-gamma. The clones FA-28 and BG-39, which failed to induce IgE synthesis, produced, compared with the other clones tested, relatively high quantities of IFN-gamma (4700 and 2500 pg/ml, respectively). These high levels of IFN-gamma accounted for the lack of induction of IgE synthesis, because in the presence of a polyclonal anti-IFN-gamma antiserum, supernatants of FA-10 and BG-39 induced significant IgE production. In addition, the low degree of IgE production induced by supernatants of two other T cell clones (FA28 and BG24) was 15- and 3-fold enhanced, respectively, in the presence of the anti-IFN-gamma antiserum. IgE synthesis by normal B cells was also induced by rIL-4, indicating that IL-4 present in T cell clone supernatants was responsible for induction of IgE production. This notion was supported by the finding that IgE production induced by supernatant of BG-24 was strongly inhibited by a polyclonal anti-IL-4 antiserum. In contrast, IgG and IgA production induced by supernatant of BG-24 were not significantly affected by the anti-IL-4 antiserum. Only a slight inhibition of IgM synthesis was observed. Collectively, our results indicate that both recombinant and naturally produced IL-4 induce normal human B cells to synthesize IgE. However, final IgE production induced by T cell clone supernatants is the net result of the inducing and suppressive effects of IL-4 and IFN-gamma respectively, that are secreted simultaneously by the T cell clones upon activation.     

Recognition of viral antigens by human influenza A virus-specific T lymphocyte clones

The nature of the viral antigens recognized by influenza A virus-immune cytotoxic T lymphocytes (CTL) is still a matter of debate. We have used four human influenza A virus-specific T lymphocyte clones with antigen-specific cytotoxic and proliferative activity to investigate the requirements for recognition of viral antigens on infected cells. One clone recognized a cross-reactive determinant on the viral hemagglutinin, and two clones were specific for different epitopes on the viral nucleoprotein (NP). A fourth clone seemed to be specific for the viral M protein. Target cell recognition was abrogated by the addition, during infection, of the lysosomotropic drug chloroquine, known to inhibit antigen processing. Furthermore, target cells that had been pulsed with soluble purified NP were recognized and were lysed by the NP-specific clone. This reaction could also be abrogated by the addition of chloroquine during pulsing. These results were obtained irrespective of whether EBV-transformed B lymphoblastoid cells or Ia antigen-expressing T cell blasts were used as target cells. It is concluded that CTL can recognize internal viral proteins that are actively presented at the surface of the target cell. These data indicate that probably every viral protein can function as a target molecule for virus-immune CTL.     

Structural and functional studies of HLA-DR restricted antigen recognition by human helper T lymphocyte clones by using transfected murine cell lines

Murine L cells expressing the products of transfected HLA-DR1 genes functioned as APC for two influenza-specific, human Th cell clones with comparable efficiency to a DR1-expressing human lymphoblastoid cell line. In order to investigate the restriction specificity of the two Th clones, a transfectant expressing the species-mismatched MHC class II dimer DR1:I-E was tested as an APC. Both T cells showed no loss of Ag sensitivity due to substitution of the murine chain. One of the Th clones, TLC 72, showed even greater degeneracy by responding to Ag in the context of I-Ek. Taking into account the lower level of MHC class II expression on the I-Ek transfectant, there is remarkably little loss of efficiency of Ag-induced T cell activation due to the substitution of I-E for DR as restriction element. The Ag-specific responses of both clones were inhibited by anti-CD4 antibody when DR-transfected L cells or human lymphoblastoid cells were used as APC. This inhibition was also seen when Ag was presented to TLC72 by the I-Ek-expressing transfectant. Whether this inhibition is the result of negative signaling or of blocking an interaction between human CD4 and I-Ek is discussed. Similarly the inhibitory effects of mAb against the T cell accessory molecule LFA/1 were the same for both clones when either the transfectants or the lymphoblastoid cell line were used as APC, suggesting that L cells may express a molecule that is capable of acting as a ligand for human LFA/1. The results presented here further illustrate the value of transfectants in analyzing T cell recognition and accessory cell requirements. The patterns of degeneracy of MHC restriction exhibited by these clones provides a platform for a more detailed analysis of key residues involved in MHC class II-restricted T cell Ag recognition.     

A functional analysis of hematopoietic growth factor production from class I and class II human alloreactive T cell clones

Class I and Class II human alloreactive T cell clones or their conditioned media were mixed with progenitor cell-enriched null cells to assess their ability to stimulate human hematopoietic progenitor cell (HPC) growth. Optimal release of erythroid, myeloid or megakaryocyte colony-stimulating factors occurred after 72 hours and required contact of cloned T cells with irradiated stimulator cells expressing the appropriate major histocompatibility complex (MHC) determinants recognized by the T cells. Individual clones were quite heterogeneous in their capacity to release hematopoietic growth factors. Clones that produced optimal levels of factors that stimulated granulocyte-macrophage colony growth did not always produce equivalent amounts of factors that stimulated erythroid colony growth and vice versa when tested against identical target cells. Class II clones released nearly twice as much interleukin 3 activity as Class I clones. Class II clones that lacked cell-mediated lympholytic (CML) activity against B or T lymphoblastoid targets were consistent stimulators of HPC growth. In contrast, Class I or Class II clones that contained CML activity either poorly stimulated or inhibited HPC growth. These CML-positive clones produced greater amounts of gamma interferon. Our findings may have important implications for HPC growth following allogeneic mismatched bone marrow transplantation.     

The role of the T cell receptor, CD8, and LFA-1 in different stages of the cytolytic reaction mediated by alloreactive T lymphocyte clones

The cytotoxic reaction mediated by cytotoxic T lymphocytes (CTL) consists of three phases: first, the CTL binds to the target cell; next, the CTL is triggered to lyse the target cell; and in the third phase, the CTL detaches from the target cell which is lysed in the absence of the CTL. Recently, we obtained evidence that human alloreactive CTL clones initially adhere to target cells without the involvement of the interaction between the T cell receptor (Tcr) and its specific target antigen. In the present study, we investigated the effect of monoclonal antibodies specific for the Tcr on the cytotoxic reaction of three CD8+ HLA-A2-specific CTL clones, using a single cell assay in which the binding event can be distinguished from the post-binding (lytic) phase of the cytolytic reaction. It was found that monoclonal antibodies directed at a variable part of the Tcr do not affect the binding phase but strongly block the lytic phase of the cytotoxic reaction. An anti-constant region Tcr antibody and an anti-CD3 reagent had a similar effect on the two phases of the reaction as the anti-variable part Tcr antibodies. In contrast, antibodies specific for LFA-1 strongly blocked the adhesion phase but did not affect the lytic phase. Antibodies specific for CD-8 had intermediate effects. They could block both the adhesion as well as the lytic phase. The effect of anti-CD8 appeared to be dependent on the CTL clone tested. One clone was found to be inhibited in the adhesion phase, but not in the lytic phase, whereas anti-CD8 hardly blocked the adhesion phase of two other CTL clones, but affected the lytic step of those clones. Our data indicate that LFA-1 is a major adhesion molecule in the CTL reaction, whereas the Tcr/CD3 complex is implicated in a phase after the initial formation of conjugates. CD8 is associated with both steps in the cytolytic reaction. In addition to its minor role in the adhesion phase, our data suggest strongly that CD-8 is involved in the triggering phase of the cytolytic reaction.     

Soluble HLA-G inhibits cell cycle progression in human alloreactive T lymphocytes

HLA-G is involved in regulating T cell responses. Various mechanisms have been proposed to explain the inhibition of T cell proliferation. In this context, the possible role of HLA-G in cell cycle regulation remains to be explored. Using stably transfected M8 cells expressing the secreted isoform (HLA-G5) of HLA-G, we investigated the role of HLA-G in inducing apoptosis and in controlling the cell cycle of activated T cells. Soluble HLA-G (HLA-G5) inhibited both CD4 and CD8 T cell proliferation. However, HLA-G5 did not induce T cell apoptosis, as determined by 3,3'-diethyloxacarbocyanine and propidium iodine labeling. It induced accumulation of the retinoblastoma protein, but not its phosphorylated and active form. Treatment of activated T cells with HLA-G5 also reduced the amounts of cyclin D2, E, A, and B by >80%. In contrast, it induced an accumulation of p27kip, but not p21cip, in activated T cells. HLA-G does not induce apoptosis of alloreactive T cells, but induces p27kip1 and inhibits cell cycle progression.     

T cell responses to human endogenous retroviruses in HIV-1 infection

Human endogenous retroviruses (HERVs) are remnants of ancient infectious agents that have integrated into the human genome. Under normal circumstances, HERVs are functionally defective or controlled by host factors. In HIV-1-infected individuals, intracellular defense mechanisms are compromised. We hypothesized that HIV-1 infection would remove or alter controls on HERV activity. Expression of HERV could potentially stimulate a T cell response to HERV antigens, and in regions of HIV-1/HERV similarity, these T cells could be cross-reactive. We determined that the levels of HERV production in HIV-1-positive individuals exceed those of HIV-1-negative controls. To investigate the impact of HERV activity on specific immunity, we examined T cell responses to HERV peptides in 29 HIV-1-positive and 13 HIV-1-negative study participants. We report T cell responses to peptides derived from regions of HERV detected by ELISPOT analysis in the HIV-1-positive study participants. We show an inverse correlation between anti-HERV T cell responses and HIV-1 plasma viral load. In HIV-1-positive individuals, we demonstrate that HERV-specific T cells are capable of killing cells presenting their cognate peptide. These data indicate that HIV-1 infection leads to HERV expression and stimulation of a HERV-specific CD8+ T cell response. HERV-specific CD8+ T cells have characteristics consistent with an important role in the response to HIV-1 infection: a phenotype similar to that of T cells responding to an effectively controlled virus (cytomegalovirus), an inverse correlation with HIV-1 plasma viral load, and the ability to lyse cells presenting their target peptide. These characteristics suggest that elicitation of anti-HERV-specific immune responses is a novel approach to immunotherapeutic vaccination. As endogenous retroviral sequences are fixed in the human genome, they provide a stable target, and HERV-specific T cells could recognize a cell infected by any HIV-1 viral variant. HERV-specific immunity is an important new avenue for investigation in HIV-1 pathogenesis and vaccine design.     

Functional heterogeneity among human inducer T cell clones

Analysis of mouse CD4+ inducer T cells at the clonal level has established that a dichotomy among CD4+ T cell clones exists with regard to types of lymphokines secreted. Mouse T cell clones designated Th1 have been shown to secrete IL-2 and IFN-gamma, whereas T cell clones designated Th2 have been shown to produce IL-4 but not IL-2 or IFN-gamma. To determine if such a dichotomy in the helper inducer T cell subset occurred in man, we examined a panel of human CD4+ helper/inducer T cell clones for patterns of lymphokine secretion and for functional activity. We identified human T cell clones which secrete IL-4 but not IL-2 or IFN-gamma, and which appeared to correspond to murine Th2 clones. In marked contrast to murine IL-2 secreting Th1 clones which do not produce IL-4 or IFN-gamma, we observed that some human T cell clones secrete IL-2, and IFN-gamma as well as IL-4. Southern blot analysis indicated that these multi-lymphokine-secreting clones represented the progeny of a single T cell. IL-4 secretion did not always correlated with enhanced ability to induce Ig synthesis. Although one T cell clone which secreted IL-2, IL-4, and IFN-gamma could efficiently induce Ig synthesis, another expressed potent cytolytic and growth inhibitory activity for B cells, and was ineffective or inhibitory in inducing Ig synthesis. These results indicate that although the equivalent of murine Th2 type cells appears to be present in man, the simple division of T cells into a Th1 and Th2 dichotomy may not hold true for human T cells.     

Interleukin-10 containing normal human serum inhibits granzyme B release but not perforin release from alloreactive and EBV-specific T cell clones

Interleukin-10 (IL-10), also known as cytokine synthesis inhibitory factor, is capable of inhibiting synthesis of pro-inflammatory cytokines like IFNγ, IL-2, IL-3, TNFα and GM-CSF made by cells such as macrophages and T helper Type 1 cells. We observed that normal human serum, derived from a healthy individual but containing large amounts of IL-10, inhibited cytotoxic activity and interfered with granzyme B release from alloreactive cytotoxic T cell (CTL) clones in vitro, but did not affect perforin release. The addition of normal human serum containing high levels of anti-IL-10 IgG neutralized the inhibitory effects of IL-10 serum. Moreover, we have identified that cytotoxic activity and granzyme B release from an Epstein-Barr virus (EBV)-specific CTL clone was similarly inhibited in the presence of IL-10 serum, while perforin release was unaffected. Anti-IL-10 IgG serum also appeared to neutralize the inhibitory effect of IL-10 serum on an EBV-specific CTL clone.